Rapoport A P, Luhowskyj S, Doshi P, DiPersio J F
Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY, USA.
Blood. 1996 Jan 1;87(1):112-22.
The alpha subunit of the human interleukin-3 receptor (IL-3R alpha) is a 70-kD glycoprotein member of the hematopoietin receptor superfamily. This protein associates with a beta subunit common to the receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to form a high-affinity receptor for IL-3. To identify regions of IL-3R alpha critical for ligand binding and receptor function, cDNAs encoding mutant receptors were generated and expressed in COS cells along with the beta subunit. Mutant receptors lacking almost the entire cytoplasmic domain of IL-3R alpha [IL-3R alpha(CD)] or carrying a substitution of trp for leu in the membrane proximal leu-ser-x-trp-ser (LSXWS) box bound 125I-IL-3 with nearly the same affinity as wild-type IL-3R alpha. In contrast, a mutant lacking the entire "LSXWS" motif failed to bind 125I-IL-3 with high affinity despite showing surface expression. In addition, hybrid receptors composed of the first 104 amino acids (aa) of IL-3R alpha joined to aa 118 through 400 of the alpha subunit of the GM-CSF receptor (GM-R alpha) [IL-3R alpha/GM-R alpha] or the first 118 aa of GM-R alpha joined to aa 104 through 378 of IL-3R alpha [GM-R alpha/IL-3R alpha] failed to bind 125I-IL-3 in the presence of the beta subunit. A third hybrid receptor composed of the first 281 residues of IL-3R alpha fused to residues 306 through 379 of GM-R alpha [IL-3R alpha/GM-R alpha-DS] also failed to bind 125I-IL-3 in the presence of the beta subunit but, in contrast to the IL-3R alpha/GM-R alpha hybrid, demonstrated weak surface expression. Mutant receptors lacking the N-terminal 30 aa and the N-terminal 9 aa also did not bind 125I-IL-3 with high affinity, although both were expressed on the cell surface. These data suggest that although the cytoplasmic domain and the leucine residue of the "LSXWS" box are not critical for ligand binding or beta-subunit association, the "LSXWS" motif and amino-terminal sequences are important for these functions.
人白细胞介素-3受体（IL-3Rα）的α亚基是造血受体超家族的一个70-kD糖蛋白成员。该蛋白与IL-5和粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体共有的β亚基结合，形成IL-3的高亲和力受体。为了确定对配体结合和受体功能至关重要的IL-3Rα区域，生成了编码突变受体的cDNA，并与β亚基一起在COS细胞中表达。几乎缺失IL-3Rα整个胞质结构域的突变受体[IL-3Rα(CD)]或在膜近端亮氨酸-丝氨酸-x-色氨酸-丝氨酸（LSXWS）框中携带色氨酸替代亮氨酸的突变受体，与野生型IL-3Rα结合125I-IL-3的亲和力几乎相同。相比之下，一个缺失整个“LSXWS”基序的突变体尽管有表面表达，但未能以高亲和力结合125I-IL-3。此外，由IL-3Rα的前104个氨基酸（aa）与GM-CSF受体（GM-Rα）的α亚基的第118至400个aa连接组成的杂合受体[IL-3Rα/GM-Rα]，或由GM-Rα的前118个aa与IL-3Rα的第104至378个aa连接组成的杂合受体[GM-Rα/IL-3Rα]，在有β亚基存在时未能结合125I-IL-3。第三个由IL-3Rα的前281个残基与GM-Rα的第306至379个残基融合组成的杂合受体[IL-3Rα/GM-Rα-DS]，在有β亚基存在时也未能结合125I-IL-3，但与IL-3Rα/GM-Rα杂合受体不同的是，表现出较弱的表面表达。缺失N端30个aa和N端9个aa的突变受体也不能以高亲和力结合125I-IL-3，尽管两者都在细胞表面表达。这些数据表明，尽管胞质结构域和“LSXWS”框的亮氨酸残基对配体结合或β亚基结合不是关键的，但“LSXWS”基序和氨基末端序列对这些功能很重要。
